Click on image to view larger version.
Figure 4. CNR1 binding to dental epithelial and mesenchymal cells. (A) Schematic structure of CNR1 and tagged CNR1 fusion proteins and their cell-binding activity. Recombinant proteins were incubated with cells, and bound proteins were analyzed by staining with anti-Hisx6 or anti-HA antibody, with FITC-conjugated secondary antibody. Activity of CNR binding to dental epithelial cells and mesenchymal cells is shown as + (positive-binding cells are more than 30%) or - (positive-binding cells are 0%). The percentage of positive-binding cells is calculated as 100 x [number of FITC-positive cells] / [number of Hochest-positive cells]. S, signal peptide; EC, extracellular domain; TM, transmembrane domain; CP, cytoplasmic domain; Hisx6, polyhistidine; HA, hemaglutinin. (B) Visualized image of EC1 binding to dental epithelial or mesenchymal cells. EC1-His was incubated with cells, and bound proteins were visualized by FITC-conjugated secondary antibody. Purple signal was Hochest staining for the cell nucleus. (C) Only recombinant protein containing EC1 (lane3) bound to the dental epithelial cells, not S-His or 
EC1-His (lanes 1 and 2). EC1-HA was used to compete with EC1-His for cell binding at equal (lane 4) or 10-fold concentrations (lane 5). Bound CNR1 proteins were detected by Western blotting with anti-His antibody (upper) and anti-HA antibody (lower).
